Expression of dehydratase domains from a polyunsaturated fatty acid synthase increases the production of fatty acids in Escherichia coli

Oyola‐Robles, Delise; Rullán‐Lind, Carlos; Carballeira, Néstor M.; Baerga‐Ortiz, Abel

doi:10.1016/j.enzmictec.2013.10.010

Cited by 12 publications

(7 citation statements)

References 31 publications

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“…This postulated third mechanism is suggested by several studies. First, an E. coli recombinant expressing the DNA sequence region covering the DH domain of the P. profundum SS9 pfaC gene was shown to be capable of producing up to a 5-fold increase in TFAs over the negative control, although the dehydration reaction had not previously been identified as a limiting step in bacterial fatty acid biosynthesis [ 30 ] and overexpression of the native dehydratase from E. coli , FabA, did not increase the production of fatty acids [ 31 ]. Therefore, it was proposed that this DH domain also catalyzes the reaction of thioester hydrolysis (TE activity).…”

Section: Organization and Function Of The Pfa Gmentioning

confidence: 99%

Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use

Yoshida

Hashimoto

Hori³

et al. 2016

Marine Drugs

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The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase), the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs) such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed.

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Section: Organization and Function Of The Pfa Gmentioning

confidence: 99%

Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use

Yoshida

Hashimoto

Hori³

et al. 2016

Marine Drugs

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“…The PUFA synthase is made of three large subunits with three DH domains. One DH domain that is located in the C‐terminal region of subunit‐A shares more sequence similarity to DH domains of polyketide synthases, while the other two DH domains that reside adjacently in subunit‐C are more similar to Escherichia coli DH FabA (Oyola‐Robles et al ., 2014; Xie et al ., 2018; Hayashi et al ., 2019b). Our recent studies showed that two FabA‐like DH domains can functionally complement E .…”

Section: Introductionmentioning

confidence: 99%

Distinct functions of two FabA‐like dehydratase domains of polyunsaturated fatty acid synthase in the biosynthesis of very long‐chain polyunsaturated fatty acids

Xie

Sun

Meesapyodsuk

et al. 2020

Environmental Microbiology

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Thraustochytrium is a unicellular marine protist for the commercial production of very long-chain polyunsaturated fatty acids (VLCPUFAs). Biosynthesis of these VLCPUFAs in the protist is catalysed by a PUFA synthase comprising three subunits, each with multiple catalytic domains. Among these domains, two tandem FabA-like dehydratase domains (DH1 and DH2) in subunit-C together are responsible for introducing double bonds in VLCPUFAs. Domain swapping analysis in yeast showed that the defective phenotype of a Scfas1 mutant could be complemented by expressing an engineered ScFAS1 gene in which the DH domain was replaced by a single DH1 or mutated DH2 of the two. Heterologous expression of the PUFA synthase in E. coli showed that the mutation of DH1 of the two or deletion of DH1 or substitution of DH1 with DH2 resulted in the complete loss of activity in the biosynthesis of VLCPUFAs. Mutation of DH2 of the two or deletion of the DH2 domain produced a small amount of DPA, but not docosahexaenoic acid (DHA). These results indicate that each of the two FabA-like domains of the PUFA synthase possesses distinct function. DH1 domain is essential for the biosynthesis of VLCPUFAs, but DH2 domain is required for the biosynthesis of DHA.

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“…According to one report, an Escherichia coli strain engineered to overexpress a fragment consisting of four DH domains from the PUFA synthase enzyme complex, and the results showed that the E . coli strain expressing the DH tetradomain fragment was capable of producing up to a five-fold increase in total fatty acids over the negative control strain lacking the recombinant enzyme [38]. Cao et al engineered an efficient producer of unsaturated fatty acids by overexpressing two genes ( fabA and fabB ) associated with unsaturated fatty acid synthesis in E .…”

Section: Discussionmentioning

confidence: 99%

Cloning of the pks3 gene of Aurantiochytrium limacinum and functional study of the 3-ketoacyl-ACP reductase and dehydratase enzyme domains

et al. 2018

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Aurantiochytrium limacinum has received attention because of its abundance of polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA). DHA is synthesized through the polyketide synthase (PKS) pathway in A. limacinum. The related enzymes of the PKS pathway are mainly expressed by three gene clusters, called pks1, pks2 and pks3. In this study, the full-length pks3 gene was obtained by polymerase chain reaction amplification and Genome Walking technology. Based on a domain analysis of the deduced amino acid sequence of the pks3 gene, 3-ketoacyl-ACP reductase (KR) and dehydratase (DH) enzyme domains were identified. Herein, A. limacinum OUC168 was engineered by gene knock-in of KR and DH using the 18S rDNA sequence as the homologous recombination site. Total fatty acid contents and the degree of unsaturation of total fatty acids increased after the kr or dh gene was knocked in. The cloning and functional study of the pks3 gene of A. limacinum establishes a foundation for revealing the DHA synthetic pathway. Gene knock-in of the enzyme domain associated with PKS synthesis has the potential to provide effective recombinant strains with higher DHA content for industrial applications.

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Expression of dehydratase domains from a polyunsaturated fatty acid synthase increases the production of fatty acids in Escherichia coli

Cited by 12 publications

References 31 publications

Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use

Bacterial Long-Chain Polyunsaturated Fatty Acids: Their Biosynthetic Genes, Functions, and Practical Use

Distinct functions of two FabA‐like dehydratase domains of polyunsaturated fatty acid synthase in the biosynthesis of very long‐chain polyunsaturated fatty acids

Cloning of the pks3 gene of Aurantiochytrium limacinum and functional study of the 3-ketoacyl-ACP reductase and dehydratase enzyme domains

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